Optical resonator diagnostic device and methods of use

ABSTRACT

An implantable diagnostic device in accordance with the present disclosure provides various benefits such as a compact size thereby allowing implanting of the device inside animate objects; low cost due to incorporation of inexpensive detection circuitry and the use of conventional IC fabrication techniques; re-usability by heating thereby allowing multiple diagnostic tests to be performed without discarding the device; and a configuration that allows performing of simultaneous and/or sequential diagnostic tests for detecting one or more similar or dissimilar target molecules concurrently or at different times.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional Application61/562,308 filed on Nov. 21, 2011, entitled “Optically InterrogatedMicrocalorimeter,” which is incorporated herein by reference.

FIELD

The present teachings relate to diagnostic devices that may be used fordetecting molecules in bio-molecular bindings. More in particular, thepresent disclosure relates to an optical resonator diagnostic device andmethods of use.

BACKGROUND

Existing techniques for bio-molecular detection typically requireundesirably expensive, complicated and bulky equipment. One example of atechnique using such equipment is described in reference [1]. It isdesirable to address such handicaps in prior art diagnostic techniquesand equipment.

SUMMARY

According to a first aspect of the present disclosure, a diagnosticdevice fabricated in silicon includes: an optical resonator, an opticalwaveguide, and a detector. The optical resonator has a capture agentlocated at a binding site; the optical waveguide is configured forpropagating a laser beam, and for coupling a first portion of thepropagated laser beam into the optical resonator. The detector isconfigured for detecting: a) a first resonant wavelength generated bythe first optical resonator when no binding reaction is present at thebinding site, and b) a second resonant wavelength generated by the firstoptical resonator upon undergoing a change in refractive index when abinding reaction is present at the first binding site.

According to a second aspect of the present disclosure, a diagnosticdevice fabricated in silicon includes: an array of optical resonators,an optical waveguide, and an array of detectors. The optical resonatorhas an immunoassay site; the optical waveguide is configured forcoupling coherent light into each optical resonator in the array ofoptical resonators; and each detector in the array of detectors, whichis optically coupled to the array of optical resonators, is configuredto detect: a) a first resonant wavelength generated by a correspondingoptical resonator, and b) a second resonant wavelength generated by thecorresponding optical resonator upon undergoing a change in refractiveindex by binding of at least one molecule to the immunoassay site.

According to a third aspect of the present disclosure, a method of usinga diagnostic device includes: propagating a laser beam through anoptical waveguide of the diagnostic device; coupling at least a portionof the laser beam into a first optical resonator located in thediagnostic device, the first optical resonator comprising an immunoassaysite; detecting a first current in a first optical detector coupled tothe first optical resonator, the first current indicative of a firstresonant frequency generated by the first optical resonator in responseto the coupling of the at least a portion of the laser beam into thefirst optical resonator; and detecting a second current in the firstoptical detector, the second current indicative of an immunoassaybinding occurring at the immunoassay site, the immunoassay bindingcharacterized by a change in refractive index in the first opticalresonator and a corresponding change in the first resonant frequency.

Further aspects of the disclosure are shown in the specification,drawings and claims of the present application.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated into and constitute apart of this specification, illustrate one or more embodiments of thepresent disclosure and, together with the description of a few exampleembodiments, serve to explain the principles and implementations of thedisclosure. The components in the drawings are not necessarily drawn toscale. Instead, emphasis is placed upon clearly illustrating variousprinciples. Moreover, in the drawings, like reference numerals designatecorresponding parts throughout the several views.

FIG. 1 shows a diagnostic system that includes an implantable diagnosticdevice in accordance with a first embodiment of the present disclosure.

FIG. 2 shows an implantable diagnostic device in accordance with asecond embodiment of the present disclosure.

FIG. 3 shows some wavelength-related operational aspects pertaining tothe implantable device shown in FIG. 2.

FIG. 4 shows a graph of detector power as a function of time, whereinthe detector is part of an implantable diagnostic device in accordancewith the present disclosure.

FIG. 5 shows an implantable diagnostic device in accordance with a thirdembodiment of the present disclosure.

FIG. 6 shows an implantable diagnostic device in accordance with afourth embodiment of the present disclosure.

FIGS. 7-9 show graphs characterizing various parameters pertaining to athermal response of an implantable diagnostic device in accordance withthe present disclosure.

DETAILED DESCRIPTION

Throughout this description, embodiments and variations are describedfor the purpose of illustrating uses and implementations of theinventive concept. The illustrative description should be understood aspresenting examples of the inventive concept, rather than as limitingthe scope of the concept as disclosed herein. Furthermore, the use ofcertain words and/or phrases should be understood in the context of thedescription and it should be understood that in some instancesalternative words or phrases may be used to refer to substantiallysimilar actions or elements. As one example of such usage, it should beunderstood that phrases such as a binding site or an immunoassay sitegenerally refer to a location in an optical isolator wherein a bindingagent (referred to herein variously as a capture agent or an aptamer) isplaced in order to provide a binding mechanism for binding an object ofinterest (referred to herein variously as molecule, a foreign molecule,a target molecule, or a protein). The use of such words will beunderstood in a broad sense by persons of ordinary skill in the art andshould not be construed as limiting or exclusionary in nature. It willbe further understood that the word “implantable” is intended toindicate that the diagnostic device can be implanted inside certainobjects. However, nothing precludes the implantable diagnostic devicebeing configured and/or used in various applications outside an object.For example, the implantable diagnostic device in accordance with thedisclosure can be used for carrying out tests (such as an assay testusing a hand-held apparatus) conducted in a laboratory for purposes ofanalyzing a fluid or a liquid.

The various embodiments described herein are generally directed at adiagnostic system that includes a diagnostic device providing severaladvantages. A few examples of such advantages include: a compact sizethat allows implanting of the device inside animate objects;integrated/compact packaging that includes a low-cost detector device;manufacturability using existing silicon processes; re-usability as aresult of a controllable regeneration mechanism that allows forperforming multiple diagnostic tests without removal from an implantlocation (a human body, for example); and a configuration that allowsperforming of simultaneous and/or sequential diagnostic tests fordetecting one or more similar or dissimilar target moleculesconcurrently or at different times.

Particularly, a diagnostic device in accordance with the presentdisclosure is an implantable diagnostic device fabricated in siliconthat includes one or more lithographically defined optical sensorstogether with associated one or more low cost detectors and heaters. Theimplantable diagnostic device can be inserted into the blood stream ofan animal or a human being, for example, and operated to performlabel-free diagnostic tests without having to remove the diagnosticdevice from the animal or human being for purposes of obtaining results,or for purposes of regenerating the diagnostic device for more than onetest. These tests may be carried out with significantly more sensitivitythan conventional immunoassay and ELISA methods. These, and other,features of the implantable diagnostic device will be described below infurther detail using the various figures.

Attention is first drawn to FIG. 1, which shows a diagnostic system 100that includes an implantable diagnostic device 110 in accordance with afirst embodiment of the present disclosure. Implantable diagnosticdevice 110 includes an optical waveguide 120 for propagating a laserbeam that is injected into implantable diagnostic device 110 by acoherent light source such as laser source 105. While implantablediagnostic device 110 can be implanted into a blood stream of an animalor human being, laser source 105 is typically located outside the animalor human being. However, in certain embodiments, laser source 105 may beconfigured for insertion into the animal or human being, either as anintegrated package that contains both laser source 105 as well asimplantable diagnostic device 110; or as a separate first packagecontaining laser source 105, with the first package coupled to a secondpackage containing implantable diagnostic device 110.

A portion of the coherent light beam injected by laser source 105 intooptical waveguide 120 is diverted from the main light beam path 121 asan auxiliary light beam that is coupled into optical resonator 130 viaan auxiliary light beam path 122. The diversion may be carried out in avariety of ways. For example, in a first implementation, coupler/switch115 is a coupler that taps into the main light beam path 121 to access aportion of the light beam. In a second implementation, coupler/switch115 is an optical switch that diverts all or a portion of the coherentlight beam from main light beam path 121 into auxiliary light beam path122. Optical couplers and optical switches are known in the art, andwill not be elaborated upon herein so as to avoid detracting from theprimary focus of the present disclosure.

The coherent light beam propagated via auxiliary light beam path 122 iscoupled into optical resonator 130 where the beam is circulated (asindicated by arrow 123) in order to generate a resonant wavelength.Optical resonator 130 is shown in FIG. 1 as a circular resonator, but itshould be understood that optical resonator 130 may be implemented in avariety of ways, including resonators having a non-circular structure.

Auxiliary light beam path 122 that is coupled into optical resonator 130is directed into an optical resonant cavity, for example, a “whisperinggallery” structure (not shown) that is known in the prior art. Ingeneral, when broad spectrum light is introduced into an opticalresonant cavity, only specific wavelengths, referred to herein asresonant wavelengths, are reinforced inside the optical resonant cavityas a result of constructive interference. The resonant wavelengths aredetermined on the basis of a length of an optical path in a waveguidestructure of the optical resonant cavity (for example, a length of thepropagation path in a whispering gallery). More specifically, resonantwavelengths are determined on the basis of optical path lengthsconfigured in accordance to integer multiples of the respectivehalf-wavelengths of the resonant wavelengths.

In the present disclosure, optical resonator 130 provides for at leasttwo resonant wavelengths. The first resonant wavelength is determined bya first optical characteristic of optical resonator 130, particularly,in terms of a first optical signal path length, an absorption parameter,and/or a first refractive index of the optical signal path length. Oneor more of these parameters are defined in part by a binding site 133.Binding site 133, which is located upon an internal surface of theoptical resonant cavity of optical resonator 130, contains a captureagent 132 (an aptamer, for example). Capture agent 132 is selectivelylocated on the internal surface in a manner that facilitates a foreignmolecule 131 (alternatively referred to herein as a “target” molecule)from binding to capture agent 132. The foreign molecule 131 may be atarget molecule flowing in a blood stream of a human being (wherein thetarget molecule is of specific interest in a diagnostic test). Furtherdetails pertaining to this topic will be provided below.

The first resonant wavelength is defined when no foreign molecule 131 isbound to capture agent 132 present at binding site 133.

In contrast, a second resonant wavelength is defined when a foreignmolecule 131 is present at binding site 133. The presence of the foreignmolecule 131 at binding site 133 modifies the refractive index of thefirst optical signal path, thereby changing the first resonantwavelength to the second resonant wavelength.

The shift from the first resonant wavelength to the second resonantwavelength provides an indication that foreign molecule 131 is presentat binding site 133. In other words, implantable diagnostic device 110uses the resonant wavelength shift for detecting an occurrence of abio-molecular binding. Such a wavelength-oriented detection process notonly provides high detection sensitivity in implantable diagnosticdevice 110 but also provides additional advantages. For example,implantable diagnostic device 110 in accordance with the disclosure canbe used for re-usable, label-free bio-molecular detection in real timeor near-real time (at millisecond intervals, for example).

Implantable diagnostic device 110 further includes a detector 140,which, in contrast to expensive, complex and bulky prior art detectiondevices, can be fabricated on silicon inside the same package containingoptical resonator 130, thereby providing various advantages such ascompact size, low cost, and high detection sensitivity.

Detector 140 is basically an optical-to-electrical converter (O/Econverter) that accepts light provided out of optical resonator 130, andgenerates an electrical signal, say in the form of a detector current.More specifically, detector 140 generates a first electrical signal(say, a first detector current) in response to light provided by opticalresonator 130 at the first resonant wavelength, and generates a secondelectrical signal (say, a second detector current) in response to lightprovided by optical resonator 130 at the second resonant wavelength.

In addition to incorporating detector 140, in some implementations,implantable diagnostic device 110 incorporates a heater 125 and acalorimeter 135. Heater 125 is used to heat optical resonator 130, andmore particularly in some cases, at least a portion of optical resonator130 that houses binding site 133. Heating can be carried out for avariety of reasons. For example, heating can be carried out to detectand record a thermal response of foreign molecule 131 when bound tocapture agent 132 at binding site 133, and/or to release foreignmolecule 131 from capture agent 132 in order to prepare binding site 133to accommodate another foreign molecule 131 (of the same type, or adifferent type) as part of a subsequent diagnostic test.

When used for recording a thermal response, detector 140 provides datavia various electrical signals (for example, detector currents) thatcorrespond to various resonant wavelengths. The data may be mapped as agraph of a slope of resonance shift versus time. Since the slopeincreases with say, an antigen concentration, a standard curve can becompiled to calibrate the antigen concentration over time. The standardcurve may then be used to identify unknown concentration values based onone or more electrical signals generated in detector 140.

As pointed out above, detector 140 provides various advantages forexample, in terms of lower cost in comparison to prior art externallylocated measurement equipment, and in terms of increased efficiency andperformance as a result of integration into an implantable package inproximity to optical resonator 130.

Calorimeter 135 can be used to measure the temperature of opticalresonator 130, or more particularly in some cases, of binding site 133,when detector 140 is used to generate the various signals therebyfacilitating mapping of the graph described above. Integratingcalorimeter 135 inside implantable diagnostic device 110 providesvarious advantages, for example, in terms of lower cost in comparison toprior art externally located calorimeters, and in terms of increasedefficiency and performance as a result of being located in proximity tooptical resonator 130. However, it will be understood that in someimplementations, calorimeter 135 may not be included in its entiretyinside implantable diagnostic device 110 but may instead be locatedexternal to implantable diagnostic device 110. For example, atemperature sensor may be located inside implantable diagnostic device110 and a read-out unit may be located external to implantablediagnostic device 110. (It may also be pertinent to point out that FIG.1 does not show connectivity and access elements, such as metal tracks,wires, pins, and connectors, so as to avoid obfuscating the main focusof the disclosure).

Attention is now drawn to FIG. 2, which shows an implantable diagnosticdevice 210 in accordance with a second embodiment of the presentdisclosure. In contrast to implantable diagnostic device 110 of FIG. 1,which incorporates a single optical resonator 130, implantablediagnostic device 210 incorporates an array of optical resonators.

In a first example implementation, each of the optical resonators in thearray of optical resonators is substantially similar to opticalresonator 130 of FIG. 1. The other elements (detector, heater, andcalorimeter) are also substantially similar between the two embodiments.While only two optical resonator circuits are shown, it will beunderstood that the array of optical resonators can include “N” (N>2)number of optical resonators as indicated by the “a” through “n”suffixes in the various labels.

In a second example implementation, at least one optical resonator inthe array of optical resonators does not include a binding site having acapture agent for capturing a foreign molecule. If a single opticalresonator is configured in this manner, this single optical resonatorcan be used as a “reference” optical resonator for various measurementpurposes.

For example, in one application, a “reference” current generated in a“reference” detector (associated with a reference optical resonatorhaving no binding site) is used for analyzing one or more currentsdetected in one or more other optical resonators wherein binding sitesare provided.

In other words, the reference current, which corresponds to a firstresonant wavelength in the reference optical resonator (due to the lackof a binding site), can be compared to a first current generated in afirst optical resonator when no binding reaction is present (i.e.corresponding to a first resonant wavelength). This comparison can becarried out for example, as part of a calibration procedure.Subsequently, when a binding reaction occurs in the first opticalresonator, a second current is generated in the first optical resonator(as described above). The difference value between the first and secondcurrents in the first optical resonator can be compared to a differencevalue between the reference current (in the reference optical resonator)and the second current in the first optical resonator. As can beunderstood, if the first optical resonator was well calibrated, the tworesults will be identical. Other such comparisons between the referencecurrent and other currents generated in other optical resonators can beused for various calibration and/or measurement purposes.

FIG. 3 shows some wavelength-related operational aspects pertaining toimplantable diagnostic device 210 shown in FIG. 2. The array of opticalresonators 300 can be used for performing various types of diagnostictests such as individual diagnostic tests, concurrent tests, sequentialtests etc., which will be described below in further detail.

In a first example test procedure, each of coupler/switch 115 a-n is acoupler that directs a respective portion of light from the laser beam(not shown) propagating through optical waveguide 120. The respectiveportions of light are coupled into respective optical resonators 130 a-nand generate a first or a second resonant wavelength that is detected bythe respective detectors 140 a-n. As explained above, the first resonantwavelength is generated when no binding has occurred on a respectivebinding site 133 a-n, and the second wavelength is generated when abinding is detected at a respective binding site 133 a-n. As can beunderstood, in this example test procedure, one detector among detectors140 a-n indicates a binding event when a single binding has occurred inthe array of optical resonators 300. On the other hand, two or more ofdetectors 140 a-n indicate that two or more optical resonators 130 a-nhave binding occurrences. Such a configuration permits a concurrent testmode conducted with reference to one specific foreign molecule wherebymultiple test results obtained from detectors 140 a-n can be compared toeach other to obtain statistical data of the one specific foreignmolecule. The concurrent test mode may also be used to obtainindependent diagnostic test results for a variety of foreign molecules.In this variant of the concurrent test mode, each of binding sites 133a-n is individually functionalized with a different capture agent thatis selected for uniquely binding a corresponding foreign molecule.

In a second example test procedure, each of coupler/switch 115 a-n is anoptical switch. One of the switches is activated so as to divert all, ora portion, of light from the laser beam propagating through opticalwaveguide 120. In the example configuration shown in FIG. 2, opticalswitch 115 c is active (“on” state) while all the remaining switches areinactive (“off” state). The light diverted by optical switch 115 c iscoupled into optical resonator 130 c, wherein a first or a secondresonant wavelength is generated on the basis of a target molecule beingpresent or absent at binding site 133 c. Detector 140 c detects thegenerated first or second resonant wavelength thereby providing anindication of a binding event or absence respectively, inside opticalresonator 130 c.

In the second example test procedure described above, optical switch 115c may be selectively operated because only the diagnostic test result ofoptical resonator 130 c is desired. In a variant procedure, each ofoptical switches 115 a-n (or a sub-set of optical switches 115 a-n) maybe sequentially operated to obtain diagnostic test results from multipleoptical resonators among optical resonators 130 a-n. The sequentialoperation may conform to a particular pattern or may be carried out in arandom pattern and provides for a time-oriented diagnostic test.

Attention is next drawn to the various wavelength-related diagrams shownin FIG. 4. These wavelength-related diagrams illustrate a first examplemethod of operation wherein optical resonators 130 a-n are configuredfor operating at an offset resonant wavelength with respect to areference wavelength 312 that corresponds to a wavelength of the laserbeam propagating through optical waveguide 120. (It may be pertinent topoint out that the word “offset” may be referred to herein byalternative words such as “shifted” or “detuned.”)

Specifically, wavelength-related diagram 310 shows an offset resonantwavelength 311 that is “blue-shifted” with respect to referencewavelength 312. Offset resonant wavelength 311, as used in thisparticular method of operation, represents an absence of a binding eventat binding site 133 a (either because light is not directed throughoptical resonator 130 a, or no binding has occurred even though directedlight is present in optical resonator 130 a).

In contrast to wavelength-related diagram 310, wavelength-relateddiagram 320 not only shows an offset wavelength 321 (in dashed lineformat) but also shows a second wavelength 322. Offset wavelength 321provides the same indication as offset wavelength 311. However, secondwavelength 322 provides an indication that offset wavelength 311 hasshifted from the offset wavelength 321 position as a direct result of anoccurrence of a binding event inside optical resonator 130 c.

The wavelength shifting feature may be implemented in different ways. Ina first implementation, the wavelength shift may be designed such thatsecond wavelength 322 coincides with reference wavelength 312. In asecond implementation, the change in wavelength may be designed suchthat second wavelength 322 does not coincide with reference wavelength312.

Furthermore, depending on the nature of the testing, in one case theextent of the wavelength shift in itself is not necessarily assignificant as the fact that a change has occurred thereby signalingthat a binding event is currently taking place or has taken place priorto detection. In this case, the wavelength shift may be interpreted as abinary wavelength shift. However, in another case, the wavelength shiftis not binary in nature, and a variable amount of the wavelength shiftis used as a proportional indicator of the concentration of targetmolecules (say in a test solution, such as blood).

In a second example method of operation, each of optical resonators 130a-n is configured to have a first resonant wavelength that coincideswith reference wavelength 312 when no binding is present. When a bindingevent takes place in any one or more of optical resonators 130 a-n, thefirst resonant wavelength gets shifted to a second resonant wavelengthaway from reference wavelength 312. As described above, the shift inwavelength results from one or more foreign molecules binding to abinding site and consequently modifying the refractive index of anoptical signal path inside the particular optical resonator. Here againdepending on the nature of the testing, in one case the extent of thewavelength shift itself is not necessarily as significant as the factthat a change has occurred thereby signaling that a binding event iscurrently taking place or has taken place prior to detection. However,in another case, the extent of the wavelength shift is used as adirectly proportional indicator of the concentration of target moleculesin the tested solution (blood, for example).

In a third example method of operation, each of optical resonators 130a-n is configured to have a first resonant wavelength that coincideswith reference wavelength 312 when no binding is present. When a bindingevent takes place in any one or more of optical resonators 130 a-n, thefirst resonant wavelength gets modified to a point wherein no outputsignal is available to the corresponding detector. The modification canbe carried out in a variety of ways, such as for example, shifting thefirst resonant wavelength to a wavelength that is not detectable by thecorresponding detector, or by causing the optical resonator to stopresonating.

Any of the three example methods of operation described above can beimplemented in at least two alternative ways.

In a first implementation, which can be interpreted as constituting aconcurrent data collection process, each of coupler/switch 115 a-n(whether implemented as a coupler or as an optical switch) is activatedso as to divert all, or a portion, of light from optical waveguide 120to two or more optical resonators 130 a-n. The corresponding detectorsin detectors 140 a-n provide data indicative of the occurrence (ornon-occurrence) of a binding event in the two or more optical resonators130 a-n. The data can be retrieved sequentially or concurrently.

In a second implementation, which may be viewed as constituting a polleddata collection process, only one of coupler/switch 115 a-n (in thisembodiment, implemented as an optical switch) is activated so as todivert all, or a portion, of light from optical waveguide 120 to oneparticular optical resonator amongst optical resonators 130 a-n. Thecorresponding detector provides data indicative of the occurrence ornon-occurrence of a binding event in the particular optical resonator.Subsequently, another one of coupler/switch 115 a-n is activated anddata obtained for another optical resonator. This polling process canthen be continued for all or some of the remaining optical resonators.

Either of the two implementations described above can be carried out invarious instants in time, for example, to obtain test results over aperiod of time.

FIG. 4 shows a graph of power generated in a detector (any one ofdetectors 140 a-n) as a result of optical input provided from acorresponding optical resonator (any one of optical resonators 130 a-n)as a function of time. The change in detected power provides anindication of a binding event.

The structure, fabrication, and prepping of an implantable diagnosticdevice in accordance with the disclosure will now be described. In apreferred embodiment, the implantable diagnostic device is fabricated asan implantable module containing at least one device that is fabricatedusing integrated circuit (IC) fabrication techniques incorporatingtechniques such as lithography and optical/electron beam printing forcreating micron-sized circuitry.

For example, each of the optical resonators can be lithographicallyconstructed inside an IC by etching ring or nanobeam resonator patternsthrough thin silicon membranes supported by silicon dioxide supportlayers. Additional elements such as the detector, heater, andcalorimeter can be fabricated in one or more of the silicon supportlayers or on additional layers, or can use the geometry of the opticalresonator. Metal connections, such as interconnecting tracks, externalaccess pins, ground and power circuits etc., are then provided.

The optical resonators can be functionalized with various capture agentsto enable label-free detection of analyses, for example. For detectionof coagulants such as thrombin, chemically stable aptamer chemistrieshave been developed. The optical resonators can be functionalized withthese aptamers through a thermally assisted coating process. In such aprocess, Bis(trimethylsilyl)acetamide (BSA) or Polytetrafluoroethylene(PTFE) is first deposited onto the silicon dioxide surface of theimplantable diagnostic device. This deposition process is followed by alocal heating process of the optical resonator, during which the BSA islocally removed from the resonator, but remains on the rest of thesilicon dioxide surface. Finally, the dioxide surface is coated with thethrombin binding aptamer, which deposits only on the previously heatedresonators in which BSA was removed.

DNA immobilization procedures can be implemented by functionalizing theoptical resonators using an amino group with a spacer arm (C6) attachedto the 5′ terminus of the thrombin-binding aptamer(5′-NH2-C6-GGTTGGTGTGGTTGG-3′) and poly (dT)15. A complementary poly(dA)15 could be fluorescently labeled at the 5′ terminus usingfluorescein phosphoramidite (FAM-poly(dA)15) reporter for laboratoryconfirmation of an aptamer binding event. The aptamer immobilizationwill be covalently attachment to a silicon surface of an opticalresonator by using a silicon oxide outer layer to which silane reagentscan be conjugated. Functionalization of silanoxide optical resonatorscan be carried out using trialkoxysilane (e.g., trimethoxysilane ortriethoxysilane) reagents incorporating aminopropyl such as(3-aminopropyl) triethoxysilane (APTES) in acetone. Specifically, asalinization procedure begins with exposure of a silica surface for ˜1-2hours to a desired silane reagent (APTES) in the vapor phase or in a 5%(v/v) solution of the silane reagent in solvents such as acetone,followed by washing 3 times with acetone and drying in air or undernitrogen. The amino-silanized resonators are then immersed in 1 mM DSS(disuccinimidyl suberate) amino reactive bifunctional covalentcross-linker in DMSO for 1 hour. The substrates are then be washed withDMSO and phosphate buffered solution (PBS) (20 mM Na2HPO4, 150 mM NaCl,pH 7.4) for three times and incubated in 500 μL of 60 μMthrombin-binding aptamer in PBS or a mixture of thrombin-binding aptamercontaining poly (dT)15 and FAM-poly(dA)15 in PBS buffer for 2 h.Unreacted thrombin-binding aptamer poly (dT)15 or FAM-poly(dA)15 isremoved by washing thoroughly with a buffer solution containing 1% (w/v)BSA and 0.05% Tween-20.

A few non-limiting examples of methods of using an implantablediagnostic device in accordance with the disclosure will now bedescribed.

The first example method pertains to protein detection, and morespecifically for detecting protein concentrations in a solution. In thismethod, an implantable diagnostic device, specifically the opticalresonator, is incubated with a target protein at various concentrationsin PBS buffer solution at room temperature for 3 hours. After washingwith PBS, the resonator is rinsed thoroughly with a buffer and dried formeasurement and readout. The binding of the target protein to thebinding site results in a shift in wavelength of the optical resonatoras described above. The wavelength shift can be directly correlated tothe concentration of the target protein in the bulk solution or bloodsample. When the detector is subsequently heated, the target protein isreleased back into the blood sample, and the corresponding reduction inthe refractive index change is recorded as a shift in the resonatorwavelength. Releasing the target proteins from the binding site preparesthe implantable diagnostic device for subsequent use in testing.

Heating raises the temperature of the optical resonator so as to disturbthe interactions of the protein binding aptamer with the target protein,thereby providing non-destructive regeneration of the diagnostic devicefor multiple tests. Significantly, the regeneration can be carried outwhile the implantable diagnostic device is implanted inside a humanbody, for example, without removal from inside the human body. Aptamersare known to undergo denaturation/regeneration multiple times withoutdegradation. Another general strategy that can be used to disrupt theaptamer-protein non-covalent interactions is the 2 M NaCl asdemonstrated by Baldrich et al. [34] or the use of aqueous solutions of6 M guanidium chloride which has also been demonstrated by severalresearchers [35-38]. In case a single regeneration agent is notsufficiently effective, a combination of regeneration reagents can beused.

The second example method pertains to DNA hybridization monitoring. Inthis method, an implantable diagnostic device, specifically the opticalresonator, is functionalized with single stranded target DNA moleculesthat can serve as hybridization probes. Single-stranded DNA moleculesfrom the sample can then be matched and bonded to this functionalizedsurface, and the binding of sample to target DNA recorded as a shift inthe wavelength of the resonators. The double-stranded DNA resulting fromthis binding reaction can subsequently be heated and denatured,resulting in a “melting” curve which can be used to confirm the bindingenergy between the DNA molecules bonded to the binding site. Thisapproach of heating single-stranded molecules in the presence of anenzyme can also lead to DNA amplification at the surface of theresonator, leading to polymerase chain reaction with extremely accuratetemperature control. As only the surface of the optical resonators isheated, polymerase chain reaction (PCR) amplification can be selectivelyperformed on that surface within a room-temperature bath. The PCRreaction can also be precisely monitored by using infrared light withlittle chance of bleaching and no need for fluorescent or other labels.It should be understood that label-free detection of PCR is desirablenot only in implanted devices but in-vitro molecular diagnostic tests aswell.

The various embodiments of an implantable diagnostic device inaccordance with the disclosure can be used to conduct various othertypes of binding reactions, such as, for example, polymerase chainreaction amplification of hybridized DNA, whereby amplification of amaterial upon the binding site can be used to obtain higher sensitivitytowards, say a nucleic acid target.

FIG. 5 shows an implantable diagnostic device 500 in accordance with athird embodiment of the present disclosure. In this embodiment, in placeof a ring resonator as described above, a linear optical waveguide 510is configured to contain an array of optical resonators 515 a-d. Each ofthe resonators in the array of optical resonators 515 a-d is fabricatedby drilling a hole in linear optical waveguide 510. A proximally locatedpump/probe waveguide 505 cooperates with linear optical waveguide 510for the assembly to operate as a nanobeam resonator.

FIG. 6 shows an implantable diagnostic device 600 in accordance with afourth embodiment of the present disclosure. In contrast to the thirdembodiment, this embodiment includes a heating element 605 that can beimplemented as a metal layer mounted upon the array of opticalresonators 515 a-d. One example material that can be used for the metallayer is Ni—Cr. Contact leads 610 a-b are used to provide a suitableelectrical stimulus in order to provide heating via heating element 605.In some implementations, the electrical stimulus can be a pulsedstimulus (a pulsed current, for example) that provides controlledheating to specific areas without undesirably heating surrounding tissueor fluids. Furthermore, such a controlled heating allows surroundingtissue and/or fluids to be maintained at their intrinsic quiescenttemperature (which may be desirable in certain applications).

In an alternative implementation, heating element 605 is eliminated andcontact leads 610 a-b are directly connected to some sections (or theentire portion) of linear optical waveguide 510. The silicon material oflinear optical waveguide 510 operates as a heating component for heatingone or more of the array of optical resonators 515 a-d. The combinationof this heating component and the predictable refractive indexdispersion properties of silicon (shown in FIG. 9) not only enablesprecise thermo-optic measurements of temperature (for example, within0.01° C. accuracy), but also enables chemistry analysis of a surface andthe use of various types of micro-calorimetric procedures.

In one example application, linear optical waveguide 510 isapproximately 0.5 microns wide and 0.2 microns high, whereby opticallymeasured temperature corresponds very closely to a surface temperatureat which a binding reaction takes place.

It will be understood that though FIG. 6 indicates an array of opticalresonators 515 a-d, in certain implementations, a single opticalresonator may be used in place of the multiple resonators. Furthermore,though heating element 605 is indicated in FIG. 6 for heating all of thearray of optical resonators 515 a-d, in alternative implementations,heating element 605 can be configured for localized heating, forexample, of one individual optical resonator amongst the array ofoptical resonators 515 a-d, and more specifically for heating only aportion of an optical resonator where the binding site is located.

As indicated above, heating can be used for a number of purposes. Giventhe low mass of the heated object (for example, an individual opticalresonator), heating can be carried out in fast and flexible manner. Forexample, localized heating provides a good degree of thermal controlover a surface of an optical resonator so as to allow local chemistryand functionalization to be carried out only in specific areas whereindetection of resonant optical wavelengths is carried out. Furthermore,occasional heating of an optical resonator helps keep the opticalresonator clean by removing unwanted coatings and epithelial cellformations or bio-fouling which often plague implanted refractive indexsensors. The cleaning action also enables various other diagnostic testrelated actions, such as for example, an accurate verification of abonding energy between one or more adsorbed molecules and one or morecapture agents.

FIGS. 7-9 show graphs characterizing various parameters pertaining to athermal response of an implantable diagnostic device in accordance withthe present disclosure. In one example implementation, the implantablediagnostic device provided a thermo-electric response of 0.35 nm/mW andheating from room temperature to over 100° C. within tens ofmicroseconds.

In conclusion, an implantable diagnostic device in accordance with thepresent disclosure provides various benefits such as a compact size(thereby allowing implanting of the device inside animate objects); lowcost due to incorporation of inexpensive detection circuitry (and theuse of conventional IC fabrication techniques); re-usability by heating(thereby allowing multiple diagnostic tests to be performed withoutremoval or discarding of the device); and a configuration that allowsperforming of simultaneous and/or sequential diagnostic tests fordetecting one or more similar or dissimilar target molecules(concurrently or at different times).

The implantable diagnostic devices disclosed herein can be fabricated insilicon-on-insulator material, enabling the use of standard CMOSfabrication technology thereby providing various benefits such as lowcost and excellent manufacturability. The manufacturing can include highresolution lithography followed by anisotropic etching ofsemiconductors. In general, the fabricated implantable diagnosticdevices include membranes with perforations (nanobeam resonators asdescribed above using FIGS. 5 and 6) and geometries that incorporateplanar photonic crystal cavities. The structure and fabrication providean ability to design mode geography that ensures efficient overlapbetween an optical field and an analyte, and also provides for compactdevices having reduced mode volume.

The combination of uniform thermal control over surface temperature inthe implantable diagnostic devices disclosed herein enables theidentification of binding energies through micro-calorimetricmeasurements related to a binding test, as well as cleaning of thesurface to enable quasi-continuous testing for proteins even withincomplex intravenous chemistry. Thermal control of the optical resonatorsurface enables precise tuning of the optical resonator to the lasersource, thereby leading to simple optical feedback systems and theelimination of additional spectroscopic instrumentation. The featuresdisclosed herein that include enables the manufacture of compactinstruments that enable in-vivo continuous monitoring of severalmetabolites (proteins, DNA strands, or other interesting molecules). Themicro-calorimetric measurements enable temperature measurement with anaccuracy of approximately 0.01 C as a result of the predictable natureof the refractive index of the material from which the optical resonatoris fabricated (for example, silicon). More particularly, a thermo-opticmeasurement in accordance with the disclosure can be confined to an areaof about 100 nm. Some other materials that can be used in lieu of, or inaddition to, silicon, include transparent materials with desirablelevels of conductivity. A few non-exhaustive list of other materialsincludes: GaAs, InP, GaN, and/or combinations of InGaAsP, InGaAsN,InGaAlP or InSnO (ITO).

All patents and publications mentioned in the specification may beindicative of the levels of skill of those skilled in the art to whichthe disclosure pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

It is to be understood that the disclosure is not limited to particularmethods or systems, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. As used in this specification and the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontent clearly dictates otherwise. The term “plurality” includes two ormore referents unless the content clearly dictates otherwise. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which the disclosure pertains.

The examples set forth above are provided to give those of ordinaryskill in the art a complete disclosure and description of how to makeand use the various embodiments of the disclosure, and are not intendedto limit the scope of what the inventors regard as their disclosure.Modifications of the above-described modes for carrying out thedisclosure may be used by persons of skill in the relevant arts, and areintended to be within the scope of the following claims.

A number of embodiments of the disclosure have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the presentdisclosure. Accordingly, other embodiments are within the scope of thefollowing claims.

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What is claimed is:
 1. A diagnostic device fabricated in silicon, thediagnostic device comprising: a first optical resonator comprising acapture agent located at a binding site; an optical waveguide configuredfor propagating a laser beam and for coupling a first portion of thepropagated laser beam into the first optical resonator; and a firstdetector configured for detecting a) a first resonant wavelengthgenerated by the first optical resonator when no binding reaction ispresent at the binding site, and b) a second resonant wavelengthgenerated by the first optical resonator upon undergoing a change inrefractive index when a binding reaction is present at the first bindingsite.
 2. The diagnostic device of claim 1, wherein detecting the firstresonant wavelength comprises detecting a first current in the detector,and detecting the second resonant wavelength comprises detecting achange in the first current.
 3. The diagnostic device of claim 2,wherein the optical waveguide is a linear optical waveguide and thefirst optical resonator is a linear nanobeam resonator comprising one ormore holes in the linear optical waveguide.
 4. The diagnostic device ofclaim 3, wherein the diagnostic device is packaged for placement insidean animate object, the placement comprising placing the diagnosticdevice in a blood stream or in tissue close to the bloodstream of theanimate object for label-free diagnostic testing.
 5. The diagnosticdevice of claim 4, wherein the capture agent is an aptamer selected tocapture a first analyte and wherein the second resonant wavelength isshifted from the first resonant wavelength in direct proportion to aconcentration of the first analyte.
 6. The diagnostic device of claim 2,further comprising: a heating element configured for heating at least aportion of the first optical resonator.
 7. The diagnostic device ofclaim 6, wherein the linear optical waveguide is configured at least inpart, to be operative as the heating element.
 8. The diagnostic deviceof claim 6, further comprising: a calorimeter for measuring atemperature of the first binding site.
 9. The diagnostic device of claim8, wherein the calorimeter is an integral part of the first opticalresonator.
 10. The diagnostic device of claim 9, wherein the calorimeteremploys a thermo-optic measurement.
 11. The diagnostic device of claim10, wherein the thermo-optic measurement provides at least one of: a) atemperature indication of a surface or b) a chemistry of the surface.12. The diagnostic device of claim 11, wherein the thermo-opticmeasurement is a micro-calorimetric measurement confinable to an area ofless than 1 micrometer in all directions.
 13. The diagnostic device ofclaim 2, further comprising: a second optical resonator comprising asecond binding site, the second optical resonator configured to receivea second portion of the laser beam propagated through the opticalwaveguide; and a second detector configured for detecting a) a thirdresonant wavelength generated by the second optical resonator when nobinding reaction is present at the second binding site, and b) a fourthresonant wavelength generated by the second optical resonator uponundergoing a change in refractive index when a binding reaction ispresent at the second binding site.
 14. The diagnostic device of claim13, wherein the third resonant wavelength is the same as the firstresonant wavelength, and the fourth resonant wavelength is the same asthe second resonant wavelength.
 15. A diagnostic device fabricated insilicon, the diagnostic device comprising: an array of opticalresonators, each optical resonator comprising an immunoassay site; anoptical waveguide configured for coupling coherent light into one ormore optical resonators in the array of optical resonators; and an arrayof detectors optically coupled to the array of optical resonators, eachdetector in the array of detectors configured to detect: a) a firstresonant wavelength generated by a corresponding optical resonator, andb) a second resonant wavelength generated by the corresponding opticalresonator upon undergoing a change in refractive index by binding of atleast one molecule to the immunoassay site.
 16. The diagnostic device ofclaim 15, wherein each detector generates a first current in response tothe first resonant wavelength and a second current in response to thesecond resonant wavelength.
 17. The diagnostic device of claim 16,wherein the first resonant wavelength is selected to substantiallycoincide with a reference wavelength that is associated with the laserbeam propagating through optical waveguide.
 18. The diagnostic device ofclaim 16, wherein the first resonant wavelength is offset with respectto a reference wavelength associated with the laser beam propagatingthrough optical waveguide.
 19. The diagnostic device of claim 18,wherein the second current is substantially zero when the correspondingoptical resonator undergoes the change in refractive index.
 20. Thediagnostic device of claim 16, wherein the diagnostic device is packagedin a silicon-based package that is configured for placement inside ananimate object.
 21. The diagnostic device of claim 20, wherein thesilicon-based package comprises a) a silicon substrate, and b) at leastone silicon membrane in which the array of optical resonators isfabricated at least in part, by using a lithographic process.
 22. Thediagnostic device of claim 21, further comprising: a capture agentcoated on to at least a portion of the at least one silicon member toenable label-free diagnostic testing.
 23. The diagnostic device of claim16, further comprising: a plurality of heating elements configured forheating a respective one of the array of optical resonators.
 24. Thediagnostic device of claim 23, wherein the heating is directed atnon-destructively regenerating at least one of the immunoassay sites forcarrying out additional diagnostic tests.
 25. The diagnostic device ofclaim 23, wherein the heating is directed at obtaining diagnostic dataindicative of molecular concentration in a tested fluid.
 26. Thediagnostic device of claim 25, wherein the tested fluid is a bloodsample and the molecular concentration of a target protein in the bloodsample.
 27. The diagnostic device of claim 15, wherein the opticalwaveguide is a linear optical waveguide, and the array of opticalresonators comprises an array of holes in the linear optical waveguide.28. The diagnostic device of claim 27, wherein at least a portion of thelinear optical waveguide is configured as a heating element for heatingone or more of the array of optical resonators.
 29. A method of using adiagnostic device, comprising: propagating a laser beam through anoptical waveguide of the diagnostic device; coupling a first portion ofthe laser beam into a first optical resonator located in the diagnosticdevice, the first optical resonator comprising an immunoassay site;detecting a first current in a first optical detector coupled to thefirst optical resonator, the first current indicative of a firstresonant frequency generated by the first optical resonator in responseto the coupling of the first portion of the laser beam into the firstoptical resonator; and detecting a second current in the first opticaldetector, the second current indicative of an immunoassay bindingoccurring at the immunoassay site, the immunoassay binding characterizedby a change in refractive index in the first optical resonator and acorresponding change in the first resonant frequency.
 30. The method ofclaim 29, further comprising: prior to propagating the laser beamthrough the optical waveguide of the diagnostic device, incubating thefirst optical resonator with a target protein in a buffer solution atroom temperature for a first period of time; washing the first opticalresonator with the buffer solution; and drying the first opticalresonator.
 31. The method of claim 30, wherein the buffer solution is aphosphate buffered saline (PBS) solution, and the first period of timeis about 3 hours.
 32. The method of claim 31, further comprising: afterdetecting the second current indicative of the immunoassay binding,regenerating the immunoassay site for a new diagnostic test by heatingthe first optical resonator to release the target protein from theimmunoassay site.
 33. The method of claim 29, wherein the immunoassaybinding occurring at the immunoassay site is used for label-freedetection of a protein in blood.
 34. The method of claim 29, furthercomprising: prior to propagating the laser beam through the opticalwaveguide of the diagnostic device, functionalizing the first opticalresonator by addition of at least one single-stranded target DNA. 35.The method of claim 34, wherein detecting the second current in thefirst optical detector is indicative of a binding of a sample DNA to thetarget DNA to form a double-strand DNA.
 36. The method of claim 35,further comprising: heating the first optical resonator to heat anddenature the double-strand DNA for obtaining a melting curve; and usingthe melting curve to evaluate a binding energy between two or more DNAmolecules of the double-strand DNA.
 37. The method of claim 29, furthercomprising: heating the first optical resonator for at least one of: a)removing one or more contaminants or b) to minimize a reduction insensitivity of the diagnostic device over time.
 38. The method of claim29, further comprising: heating the first optical resonator anddetermining therefrom at least one of: a) a melting curve or b) atemperature at which a molecule is released from the immunoassay site.39. The method of claim 38, wherein the temperature at which themolecule is released is indicative of the identity of the molecule. 40.The method of claim 29, further comprising: heating the first opticalresonator one or more times to non-destructively regenerate theimmunoassay site after performing one or more diagnostic tests.
 41. Themethod of claim 29, further comprising: coupling a second portion of thelaser beam into a second optical resonator located in the diagnosticdevice, the second optical resonator configured to exclude immunoassaybindings; heating a portion of at least one of the first or the secondoptical resonators; detecting a third current in a second opticaldetector coupled to the second optical resonator, the third currentindicative of a third resonant frequency generated by the second opticalresonator in response to the coupling of the second portion of the laserbeam into the second optical resonator; and using the first, second andthird currents to analyze the immunoassay binding occurring at theimmunoassay site of the first optical resonator.
 42. The method of claim41, wherein using the first, second and third currents comprisescomparing at least one of the first, second and third currents toanother one of the first, second and third currents.
 43. The method ofclaim 29, further comprising: determining a temperature of a portion ofa surface of the first optical resonator by using a thermo-optic shiftcharacteristic of a material of the diagnostic device.
 44. The method ofclaim 43, wherein the material is at least one of: i) a transparentmaterial having a first electrical conductivity, ii) GaAs, iii) InP, iv)GaN, v) InGaAsP, vi) InGaAsN, vii) InGaAlP, viii) InSnO (ITO), or ix)combinations thereof.